Protein Variants Research Articles

Healthy longevity-associated protein improves cardiac function in murine models of cardiomyopathy with preserved ejection fraction

AimsAging is influenced by genetic determinants and comorbidities, among which diabetes increases the risk for heart failure with preserved ejection fraction. There is no therapy to prevent heart dysfunction in aging and diabetic individuals. In previous studies, a single administration of the longevity-associated variant (LAV) of the human BPIFB4 gene halted heart decline in older and type 2 diabetic mice. Here, we asked whether orally administered LAV-BPIFB4 protein replicates these benefits.Materials and MethodsIn two controlled, randomized studies, 18-month-old male C57BL/6 J mice and 9-week-old C57BLKS/J-Leprdb/Leprdb/Dock7 + [db/db] mice of both sexes underwent baseline echocardiography. They then received a recombinant purified LAV-BPIFB4 protein (3 µg/animal, every three days) or vehicle by gavage. After 30 days, the animals underwent echocardiography, and the hearts were collected post-termination for histology.ResultsAll the animals completed the study except one female diabetic mouse, which was culled prematurely because tooth malocclusion caused eating problems. There was no effect of the LAV-BPIFB4 protein on body weight in the two studies or glycosuria in the diabetic study. In aging mice, LAV-BPIFB4 increased myocardial Bpifb4 expression, improving heart contractility and capillarity while reducing perivascular fibrosis and senesce. In male diabetic mice, LAV-BPIFB4 therapy improved systolic function, microvascular density, and senescence, whereas the benefit was limited to systolic function in females.ConclusionsThis study shows the feasibility and efficacy of a variant protein associated with human longevity in contrasting pivotal risk factors for heart failure in animal models. The diabetic study revealed that sex influences the treatment efficacy.

The Multifunctional Anaphase Promoting Complex 7 (APC7) Gene Is Associated With Increased Plant Growth and Improved Resistance to DNA and RNA Viruses.

The anaphase promoting complex 7 (AtAPC7) is an APC/C subunit expressed in different organs of Arabidopsis thaliana and conserved among eukaryotes. A variant of the complete APC7 protein, containing its C-terminal region (named APC-CT), shows a high homology with a tobacco viral replication inhibitor (IVR-like) protein that reduces plant susceptibility to RNA viruses. Here, the role of the AtAPC7 gene was investigated by characterizing Arabidopsis plants overexpressing the full-length AtAPC7 (APC7OE) and the C-terminal portion (APC7-CTOE), by phenotypical, physiological and molecular approaches. APC7OE plants showed improved growth of vegetative organs, earlier flowering and increased photosynthetic efficiency, CO2 assimilation and productivity, compared with Col-0 control plants. Conversely, APC7-CTOE plants showed reduced susceptibility to both RNA and DNA viruses, along with an improvement in plant growth, although not surpassing APC7OE plants. Altogether, the data provide evidence for the role of the AtAPC7 in regulating cell division, expansion and differentiation, accompanied by an increase in photosynthetic capacity, resulting in enhanced plant biomass and seed yield. AtAPC7-CT might reduce growth-defence trade-offs, enabling plants to simultaneously defend themselves while promoting better growth. Our findings highlight the multifunctional role of AtAPC7, unveiling the potential of its orthologous genes as valuable biotechnological tools in important crops.

Functional significance of CYP2B6 gene rare allelic variants identified in Japanese individuals

Cytochrome P450 2B6 (CYP2B6) catalyzes the metabolism of many drugs, including efavirenz and propofol. Genetic polymorphisms in CYP2B6 alter its enzymatic activity and substantially affect its pharmacokinetics. High-frequency variants, such as CYP2B6*6, are associated with the risk of developing side effects due to reduced CYP2B6 activity. However, the impact of rare alterations on enzyme function remains unknown, and some of these variants may significantly decrease the CYP2B6 activity. Therefore, in this study, we evaluated in vitro the functional alterations in 29 missense variants of the CYP2B6 gene identified in 8,380 Japanese individuals. Wild-type CYP2B6 and 29 rare CYP2B6 variants were transiently expressed in mammalian cells. The expression levels of variant CYP2B6 proteins in the microsomal fractions extracted from 293FT cells were assessed using western blotting and reduced-carbon monoxide difference spectroscopy, and a specific peak at 450 nm was detected in the wild-type and 19 variants. Furthermore, kinetic parameters were determined by assaying the reactions with efavirenz and propofol and quantifying the metabolite concentrations. We found that 12 variants had significantly lower or abolished enzymatic activity with both the substrates. In silico three-dimensional docking and molecular-dynamics simulations suggested that these functional changes were due to conformational changes in essential regions, such as the heme-binding site and ligand channels involved in transporting substrates to the active site. These findings have implications for predicting the plasma concentrations of CYP2B6 substrates and controlling their side effects.

CRISPR-Cas9 and its Application in the Fields of Medics

Gene editing technology has become a prominent objective within the fields of medical research, the clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is one particular technology that has already been practiced in a set of clinical trials, which produced a satisfying result that have demonstrated its potential to be applied to a great range of treatments. CRISPR-Cas9 was derived from bacterial defense system, and has been applied in the field of medical research. For instance, the research practice of using CAR-T cells by CAR-T Therapy, and genetically modifying DNA strands to remove cancerous or mutated sequences. This research has great chance to promote treatments associated with genetic modification and provide new hope for patients in the future. Nevertheless, this technology is still immature, due to its potential drawbacks. Most researchers are still focusing on practices of CRISPR-Cas9 on animal models or isolated somatic cells. This article draws attention on different treatments of CRISPR-Cas9 and provides evaluation and suggestions as a reference for future practices of this technology. Overall, there are two discoveries of advancement to the CRISPR-Cas9 mechanism: modification to the guide RNA that improves the accuracy, and applying high-fidelity Cas9 protein variants to enhance its comprehensive accuracy.

Alternative Splicing: A Potential Therapeutic Target in Hematological Malignancies

Leukemia represents the most prevalent malignancy in children, constituting 30% of childhood cancer cases, with acute lymphoblastic leukemia (ALL) being particularly heterogeneous. This paper explores the role of alternative splicing in leukemia, highlighting its significance in cancer development and progression. Aberrant splicing is often driven by mutations in splicing-factor genes, which can lead to the production of variant proteins that contribute to oncogenesis. The spliceosome, a complex of small nuclear RNAs and proteins, facilitates RNA splicing, a process critical for generating diverse mRNA and protein products from single genes. Mutations in splicing factors, such as U2AF1, SF3B1, SRSF2, ZRSR2, and HNRNPH1, are frequently observed across various hematological malignancies and are associated with poor prognosis and treatment resistance. This research underscores the necessity of understanding the mechanisms of RNA splicing dysregulation in order to develop targeted therapies to correct these aberrant processes, thereby improving outcomes for patients with leukemia and related disorders.

Regulation of HTT mRNA Biogenesis: The Norm and Pathology

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in exon 1 of the HTT gene, leading to the formation of a toxic variant of the huntingtin protein. It is a rare but severe hereditary disease for which no effective treatment method has been found yet. The primary therapeutic targets include the mutant protein and the mutant mRNA of HTT. Current clinical trial approaches in gene therapy involve the application of splice modulation, siRNA, or antisense oligonucleotides for RNA-targeted knockdown of HTT. However, these approaches do not take into account the diversity of HTT transcript isoforms in the normal conditions and in HD. In this review, we discuss the features of transcriptional regulation and processing that lead to the formation of various HTT mRNA variants, each of which may uniquely contribute to the progression of the disease. Furthermore, understanding the role of known transcription factors of HTT in pathology may aid in the development of potentially new therapeutic tools based on endogenous regulators.

Structural insights into the toxicity of type II ribosome inactivating proteins (RIPs): a molecular dynamics study

Ribosome Inactivating Proteins (RIPs) act by irreversibly depurinating the 28S rRNA ricin-sarcin loop (SRL) of the eukaryotic ribosome resulting in protein synthesis inhibition. In general, they consist of two variants: Type I which is single chained (∼30 kDa), and Type II, a more toxic variant which is a Type I N-glycosidase chain covalently linked to a lectin chain. These proteins are believed to play a pivotal role in defence mechanisms. Intriguingly, non-toxic variants of such toxic proteins do exist in nature. To explore their mode of action, in the present study we have selected three toxic (Ricin, Ebulin and HmRIP) as well as two non-toxic (BGSL and SGSL) RIPs and performed molecular docking and molecular dynamic simulations with the SRL loop. This study throws light on the structural stability and plasticity of the toxic and non-toxic RIP complexes. Furthermore, analysis of the active site cavity volume and binding free energy calculations reveal that the SRL, particularly the specific adenine (A4605), is relatively unstable in the case of non-toxic RIPs which is also supported by the free binding energy calculations, and the pocket size analysis indicates the abnormal increase in active site cavity volume of non-toxic RIPs with time. This first-of-its-kind comprehensive study of toxic and non-toxic RIPs gives insights about the mode of action and the dynamic nature of their interaction with the SRL loop. These observations will be helpful in the development of toxoids against RIPs and also in designing novel therapeutic approaches against human diseases.

Magnetic protein aggregates generated by supramolecular assembly of ferritin cages - a modular strategy for the immobilization of enzymes

IntroductionEfficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. This study aimed to introduce a novel immobilization strategy using in vivo-produced magnetic protein aggregates (MPAs).MethodsMPA production was achieved by expressing gene fusions of the yellow fluorescent protein variant citrine and ferritin variants, including a magnetically enhanced Escherichia coli ferritin mutant. Cellular production of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, driven by citrine-dependent dimerization of ferritin cages. Magnetic properties were confirmed using neodymium magnets. A bait/prey strategy was used to attach alcohol dehydrogenase (ADH) to the MPAs, creating catalytically active MPAs (CatMPAs). These CatMPAs were purified via magnetic columns or centrifugation.ResultsThe fusion of the mutant E. coli ferritin to citrine yielded fluorescent, insoluble protein aggregates, which are released upon cell lysis and coalesce into MPAs. MPAs display magnetic properties, as verified by their attraction to neodymium magnets. We further show that these fully in vivo-produced protein aggregates can be magnetically purified without ex vivo iron loading. Using a bait/prey strategy, MPAs were functionalized by attaching alcohol dehydrogenase post-translationally, creating catalytically active magnetic protein aggregates (CatMPAs). These CatMPAs were easily purified from crude extracts via centrifugation or magnetic columns and showed enhanced stability.DiscussionThis study presents a modular strategy for the in vivo production of MPAs as scaffold for enzyme immobilization. The approach eliminates the need for traditional, expensive carriers and simplifies the purification process by leveraging the insoluble nature and the magnetic properties of the aggregates, opening up the potential for novel, streamlined applications in biocatalysis.

A single immunization of Borreliella burgdorferi-infected mice with Vanguard crLyme elicits robust antibody responses to diverse strains and variants of outer surface protein C.

Lyme disease, caused by Borreliella burgdorferi and related species, is a growing health threat to companion animals across North America and Europe. Vaccination is an important preventive tool used widely in dogs living in, or near, endemic regions. In this report, we assessed anti-outer surface protein (Osp) A and anti-OspC antibody responses in B. burgdorferi-infected and -naïve mice (C3H/HeN) after immunization with a murine-optimized single dose of the Lyme disease subunit vaccine, Vanguard crLyme. crLyme is comprised of OspA and an OspC chimeritope-based immunogen designated as CH14. Mice that were infected and immunized developed higher levels of anti-OspC antibodies (Abs) than those infected only or that received one vaccine dose. The anti-OspC Abs that developed in the infected/immunized mice bound to all OspC variants tested (n = 22), whereas OspC Abs in serum from infected mice bound predominantly to the OspC variant (type A) produced by the infecting B. burgdorferi strain. Consistent with the absence of OspA expression in infected mammals, none of the infected mice developed Abs to OspA and did not develop anti-OspA Abs after single dose immunization. Lastly, serum from infected/immunized mice displayed significantly higher and broader killing activity than serum from non-immunized infected mice. The results of this study demonstrate that a single vaccination of actively infected mice results in strong anti-OspC Ab responses. This study contributes to our understanding of Ab responses to vaccination in actively infected mammals.

Diversity, functional classification and genotyping of SHV β-lactamases in Klebsiella pneumoniae.

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla SHV alleles but no other acquired β-lactamases to assess genotype-phenotype relationships for bla SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq.

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.

Genetic variation in bovine milk proteins: Implications for functional and nutritional properties

The six major bovine milk proteins are encoded by highly polymorphic chromosomes. Genetic variation has become a topic of interest and controversy in recent years due to supposed adverse health effects, beginning with the A2 variant of β‐casein. The functional implications of genetic variants have been well characterised, specifically heat stability and coagulation (i.e. rennet and acid) properties of milk, which have found that the B variant of β‐casein, κ‐casein and β‐lactoglobulin are the most beneficial. Genetic variants of milk proteins provide considerable potential to create new dairy products to add value to milk.

Base editing screens define the genetic landscape of cancer drug resistance mechanisms.

Drug resistance is a principal limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can retrospectively delineate the genetic basis of drug resistance, but this requires large numbers of post-treatment samples to nominate causal variants. Here we prospectively identify genetic mechanisms of resistance to ten oncology drugs from CRISPR base editing mutagenesis screens in four cancer cell lines using a guide RNA library predicted to install 32,476 variants in 11 cancer genes. We identify four functional classes of protein variants modulating drug sensitivity and use single-cell transcriptomics to reveal how these variants operate through distinct mechanisms, including eliciting a drug-addicted cell state. We identify variants that can be targeted with alternative inhibitors to overcome resistance and functionally validate an epidermal growth factor receptor (EGFR) variant that sensitizes lung cancer cells to EGFR inhibitors. Our variant-to-function map has implications for patient stratification, therapy combinations and drug scheduling in cancer treatment.

Improving CsOAC Activity in Saccharomyces cerevisiae for directed Production of Olivetolic Acid through Rational Design.

Olivetolic acid (OA) is an essential precursor in the cannabinoid biosynthesis. It is produced through a unique interaction between the two proteins, olivetol synthase (CsOLS) and olivetolic acid cyclase (CsOAC). When the OA biosynthesis is transferred to Saccharomyces cerevisiae, olivetol (OL) is produced as a side product, even with a high enhancement of copy number of CsOAC. In order to increase the OA titer while decreasing the OL titer in S. cerevisiae, rational design was applied to CsOAC using in silico approaches such as protein-ligand docking to find potential protein variants. In vivo screening and also testing different approaches for both proteins was applied to identify the best performing variants of CsOAC. Four variants were identified that gave the desired properties. The best CsOAC variant, G82A/L92Y, resulted in a 1.7-fold increase in OA production and a shift in the ratio between the two products towards OA.

Re-appraising the evidence for the source, regulation and function of p53-family isoforms.

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

Robotic microinjection enables large-scale transgenic studies of Caenorhabditis elegans

The nematode Caenorhabditis elegans is widely employed as a model organism to study basic biological mechanisms. However, transgenic C. elegans are generated by manual injection, which remains low-throughput and labor-intensive, limiting the scope of approaches benefitting from large-scale transgenesis. Here, we report a robotic microinjection system, integrating a microfluidic device capable of reliable worm immobilization, transfer, and rotation, for high-speed injection of C. elegans. The robotic system provides an injection speed 2-3 times faster than that of experts with 7–22 years of experience while maintaining comparable injection quality and only limited trials needed by users to become proficient. We further employ our system in a large-scale reverse genetic screen using multiplexed alternative splicing reporters, and find that the TDP-1 RNA-binding protein regulates alternative splicing of zoo-1 mRNA, which encodes variants of the zonula occludens tight junction proteins. With its high speed, high accuracy, and high efficiency in worm injection, this robotic system shows great potential for high-throughput transgenic studies of C. elegans.

Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.

High-Throughput Hybridization Assay as First-Line Diagnostic Test for Sarcomas: Clinical Assessment in a Tertiary Referral Center.

Sarcomas are rare and highly heterogeneous mesenchymal tumors with deceptive morphologic features that pose a challenge for precise diagnostics. Chromosomal rearrangements generating pathognomonic gene fusions are useful diagnostic markers, traditionally tested using single-plex standard of care assays with limited diagnostic yield. NanoString nCounter technology has emerged as a robust solution with multiplexing capabilities. To optimize NanoString effective coverage of specific entities and conduct a validation study to support its clinical implementation. We reconfigured a NanoString's codeset by including a set of probes for detecting gene fusion variants of solitary fibrous tumors, low-grade fibromyxoid sarcomas/sclerosing epithelioid fibrosarcomas, and undifferentiated small round cell sarcomas, totaling 188 probes. A technical validation study was conducted with 96 retrospective samples. Additionally, 76 prospective samples were evaluated to assess the assay's clinical performance. Both technical and clinical validation studies showed that NanoString's codeset reached >88% sensitivity and 100% specificity, compared with standard of care methods, and superior diagnostic yield as a first-line test. Our design enabled the detection of almost all fusion variants of NGFI-A binding protein 2 (NAB2) with signal transducer and activator of transcription 6 (STAT6) in solitary fibrous tumors, as well as cAMP responsive element binding protein 3 like 1/2 (CREB3L1/2) rearrangements in all low-grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma cases. Identification of specific gene fusions of undifferentiated small round cell sarcoma was also improved, but additional strategies are necessary to attain full coverage. The NanoString platform demonstrated good sensitivity, specificity, and superior diagnostic yield. It is a cost-effective assay with rapid turnaround time, low sample consumption, streamlined analysis, and easy customization. Therefore, it is a promising alternative first-line diagnostic tool for routine sarcoma testing.

6429 An Unusual Gene Mutation and Clinical Manifestation of MODY 10

Abstract Disclosure: J. Ambalavanan: None. O. Abdulhussein: None. J. Ferri-Guerra: None. G. Madrigal Loria: None. K. Alkwatli: None. A. Iqbal: None. R.R. Correa: None. Introduction: Maturity-Onset Diabetes of the Young (MODY) is a rare monogenic form of diabetes characterized by impaired beta cell function accounting for < 5% of all people with diabetes. About 14 different subtypes of MODY have been described in the. MODY 10 is an extremely rare form with mutation in the INS (insulin) gene. We present a previously unreported genetic mutation in the INS gene, its clinical course and subsequent management. Case: A 23-year-old female presented to us for consultation regarding new diagnosis of diabetes. She came to medical attention for pre-diabetes at the age of 16 years and was managed with diet control. She was officially diagnosed with diabetes by PCP in October 2022 after onset of polyuria and nocturia, and HbA1c of 8.7%. During initial evaluation, we suspected type 1 diabetes due to her age, low BMI of 17.61 kg/m2 and prescribed a CGM while we awaited extensive antibody evaluation. Within a week she reported average blood sugars in the 200-300 mg/dl range. As a result, she was on insulin glargine 5 units at bedtime. Within 3 months, her HbA1c dropped to 6.5% but she started experiencing frequent symptomatic hypoglycemia at work. At this point we suspected MODY due to age at diagnosis; antibody negative status for GAD-65, IA-2, ZnT8 and insulin; detectable C-peptide 1.7 ng/ml (0.81-3.85); low BMI and good glycemic control and frequent hypoglycemia on insulin. On evaluation of family history, she had a history of diabetes in maternal grandmother, paternal grandfather and paternal great uncle. Her parents did not have diabetes. We stopped glargine and started glipizide XL 2.5mg once daily. She was also referred to our clinical genetics team. On genetic evaluation she was found to have one heterozygous variant of uncertain significance in the INS gene. On further evaluation, this was found to be guanine to adenine substitution at c:139 in exon 2 of the INS gene on chromosome 11:2182063 resulting in a variant protein p.Gly47Ser which is a missense change that occurs when glycine (Gly) is replaced by serine (Ser) at position 47. This mutation has not been previously reported to be pathogenic or benign and has not been reported in significant frequency anywhere based on our literature search. Furthermore, in silico analysis supported that this missense variant had a deleterious effect on the insulin structure/function. Given the rarity of this condition, there are no formal guidelines or consensus regarding the management. During a subsequent follow up visit, we discussed that she fit the diagnosis clinically and suggested possible medications. In the end, we decided to change her from glipizide to sitagliptin 50mg daily as she was continuing to have lows at work. This helped avoid hypoglycemia and maintained time in rage at 98%. Conclusion: We present a patient with rare form of MODY 10 with a new mutation in c139:G>A at exon 2 of INS gene managed with sulfonylurea and eventually DPP4 inhibitor. Presentation: 6/2/2024

Quantitative Assessment of Conformational Heterogeneity in Apolipoprotein E4 Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Apolipoprotein E4 (apoE4) is the strongest genetic risk factor for Alzheimer's disease (AD). However, structural differences between apoE4 and the AD-neutral isoform, apoE3, still remain unclear. Recent studies suggest that apoE4 harbors intermediates. However, the biophysical properties and isoform specificity of these intermediates are not known. Here, we use the kinetics of hydrogen-deuterium exchange by mass spectrometry (HDX-MS) to examine the conformational heterogeneities in apoE3 and apoE4. First, we use numerical simulations to compute the HDX-mass spectra of a protein following mixed EX1/EX2 kinetics. The results indicate that in the presence of EX1 kinetics, which is an indicator of conformational heterogeneity, time evolution of the standard deviation (σ(t)) of the spectra exhibits a clear peak, which is dependent on the number of residues (NEX1) and the rate constant of EX1 kinetics (kEX1). Then, we performed experiments with several variants of the apoE proteins and compared them with simulation to estimate NEX1 and kEX1. Kinetics of the mean deuteration is found to be faster for apoE4, consistent with its poorer stability than apoE3. Importantly, in the case of apoE4, σ(t) exhibits a clear peak at t ∼ 60 s, but apoE3 shows only a small peak at 1800 s. Therefore, both NEX1 and kEX1 are larger for apoE4, indicating greater conformational heterogeneity. Notably, the partial EX1 kinetics is observed in both the isolated N-terminal fragment and the full-length form of apoE4, although it is more pronounced in the full-length protein. Moreover, it is enhanced at higher pH and in the presence of bis-ANS. Mutations such as R61T and R112I diminish the EX1 kinetics, making apoE4 behave more like apoE3. Thus, the amino acid substitution at position 112 alters the structural dynamics of the N-terminal domain of apoE4; the changes are further propagated and amplified in the full-length protein. We conclude that HDX-MS is a label-free and robust methodology to characterize structural heterogeneities of proteins even under native conditions. This opens opportunities for screening of the "structure corrector" drug molecules that could convert apoE4 to apoE3-like.